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FHL2 expression and variants in hypertrophic cardiomyopathy.

Friedrich FW, Reischmann S, Schwalm A, Unger A, Ramanujam D, Münch J, Müller OJ, Hengstenberg C, Galve E, Charron P, Linke WA, Engelhardt S, Patten M, Richard P, van der Velden J, Eschenhagen T, Isnard R, Carrier L - Basic Res. Cardiol. (2014)

Bottom Line: HCM is a myocardial disease characterized by left ventricular hypertrophy, diastolic dysfunction and increased interstitial fibrosis and is mainly caused by mutations in genes coding for sarcomeric proteins.We assessed the structural and functional consequences of the nonsynonymous substitutions after adeno-associated viral-mediated gene transfer in cardiac myocytes and in 3D-engineered heart tissue (EHT).Finally, chronic phenylephrine stimulation depressed EHT function in all groups, but to a lower extent in T171M-transduced EHTs.

View Article: PubMed Central - PubMed

Affiliation: Department of Experimental Pharmacology and Toxicology, Cardiovascular Research Center, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.

ABSTRACT
Based on evidence that FHL2 (four and a half LIM domains protein 2) negatively regulates cardiac hypertrophy we tested whether FHL2 altered expression or variants could be associated with hypertrophic cardiomyopathy (HCM). HCM is a myocardial disease characterized by left ventricular hypertrophy, diastolic dysfunction and increased interstitial fibrosis and is mainly caused by mutations in genes coding for sarcomeric proteins. FHL2 mRNA level, FHL2 protein level and I-band-binding density were lower in HCM patients than control individuals. Screening of 121 HCM patients without mutations in established disease genes identified 2 novel (T171M, V187L) and 4 known (R177Q, N226N, D268D, P273P) FHL2 variants in unrelated HCM families. We assessed the structural and functional consequences of the nonsynonymous substitutions after adeno-associated viral-mediated gene transfer in cardiac myocytes and in 3D-engineered heart tissue (EHT). Overexpression of FHL2 wild type or nonsynonymous substitutions in cardiac myocytes markedly down-regulated α-skeletal actin and partially blunted hypertrophy induced by phenylephrine or endothelin-1. After gene transfer in EHTs, force and velocity of both contraction and relaxation were higher with T171M and V187L FHL2 variants than wild type under basal conditions. Finally, chronic phenylephrine stimulation depressed EHT function in all groups, but to a lower extent in T171M-transduced EHTs. These data suggest that (1) FHL2 is down-regulated in HCM, (2) both FHL2 wild type and variants partially protected phenylephrine- or endothelin-1-induced hypertrophy in cardiac myocytes, and (3) FHL2 T171M and V187L nonsynonymous variants induced altered EHT contractility. These findings provide evidence that the 2 novel FHL2 variants could increase cardiac function in HCM.

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FHL2 expression and sarcomeric localization in human and mouse hearts. FHL2 mRNA level in human (a) and mouse (b, c) HCM and normal hearts (n = 8 for human, n = 4–8 for mouse). Data are expressed as mean ± SEM. *p < 0.05 vs. healthy controls/WT, unpaired Student’s t test (in a and b) or one-way ANOVA with Dunnett’s multiple comparison vs WT (in c). d–e FHL2 protein level in human HCM and control hearts. d Representative Western blot stained with an antibody recognizing FHL2 and with an antibody directed against calsequestrin, used as an endogenous control. Molecular weight markers (MW) indicate 25 and 55 kDa. e Protein levels of FHL2, normalized to calsequestrin levels and indexed to protein levels of healthy controls. Data are expressed as mean ± SEM. ***p < 0.001 vs. healthy controls, unpaired Student’s t test (in e). f–l Immunoelectron microscopy using an anti-FHL2 antibody in control and disease cardiac sections showing a specific labeling pattern in the I-band (zoom-in right upper corner of each image) of the sarcomeres; f human donor heart; g patient with HCM; h patient with aortic stenosis; i patient with obstructive HCM; j WT mouse; kMybpc3-targeted knock-in mouse. Scale bars 1 µm.  AoS aortic stenosis, Hom-KI homozygous Mybpc3-targeted knock-in mice, Het-KO heterozygous Mybpc3-targeted knock-out, Hom-KO homozygous Mybpc3-targeted knock-out mice, HOCM obstructive HCM
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Fig1: FHL2 expression and sarcomeric localization in human and mouse hearts. FHL2 mRNA level in human (a) and mouse (b, c) HCM and normal hearts (n = 8 for human, n = 4–8 for mouse). Data are expressed as mean ± SEM. *p < 0.05 vs. healthy controls/WT, unpaired Student’s t test (in a and b) or one-way ANOVA with Dunnett’s multiple comparison vs WT (in c). d–e FHL2 protein level in human HCM and control hearts. d Representative Western blot stained with an antibody recognizing FHL2 and with an antibody directed against calsequestrin, used as an endogenous control. Molecular weight markers (MW) indicate 25 and 55 kDa. e Protein levels of FHL2, normalized to calsequestrin levels and indexed to protein levels of healthy controls. Data are expressed as mean ± SEM. ***p < 0.001 vs. healthy controls, unpaired Student’s t test (in e). f–l Immunoelectron microscopy using an anti-FHL2 antibody in control and disease cardiac sections showing a specific labeling pattern in the I-band (zoom-in right upper corner of each image) of the sarcomeres; f human donor heart; g patient with HCM; h patient with aortic stenosis; i patient with obstructive HCM; j WT mouse; kMybpc3-targeted knock-in mouse. Scale bars 1 µm.  AoS aortic stenosis, Hom-KI homozygous Mybpc3-targeted knock-in mice, Het-KO heterozygous Mybpc3-targeted knock-out, Hom-KO homozygous Mybpc3-targeted knock-out mice, HOCM obstructive HCM

Mentions: It has been shown that FHL2 protein level is markedly reduced in human failing hearts [4]. To examine whether FHL2 expression is altered in HCM we measured both mRNA and protein levels in ventricular tissue of HCM patients carrying MYBPC3 mutations [52]. FHL2 mRNA and FHL2 protein levels were >50 % lower in HCM patients than in controls (Fig. 1a, d, e). Similarly, FHL2 mRNA levels were markedly reduced in two HCM mouse models, the homozygous Mybpc3-targeted knock-in (Hom-KI) [59], and both heterozygous (Het-KO) and homozygous (Hom-KO) Mybpc3-targeted knock-out mice (Fig. 1b, c; [6]). Moreover, whereas no difference was observed in the right ventricle between WT, Het-KO and Hom-KO, FHL2 mRNA level was lower merely in the hypertrophied parts of the Het-KO (=septum) and Hom-KO (=septum + LV; Online Fig. 1).Fig. 1


FHL2 expression and variants in hypertrophic cardiomyopathy.

Friedrich FW, Reischmann S, Schwalm A, Unger A, Ramanujam D, Münch J, Müller OJ, Hengstenberg C, Galve E, Charron P, Linke WA, Engelhardt S, Patten M, Richard P, van der Velden J, Eschenhagen T, Isnard R, Carrier L - Basic Res. Cardiol. (2014)

FHL2 expression and sarcomeric localization in human and mouse hearts. FHL2 mRNA level in human (a) and mouse (b, c) HCM and normal hearts (n = 8 for human, n = 4–8 for mouse). Data are expressed as mean ± SEM. *p < 0.05 vs. healthy controls/WT, unpaired Student’s t test (in a and b) or one-way ANOVA with Dunnett’s multiple comparison vs WT (in c). d–e FHL2 protein level in human HCM and control hearts. d Representative Western blot stained with an antibody recognizing FHL2 and with an antibody directed against calsequestrin, used as an endogenous control. Molecular weight markers (MW) indicate 25 and 55 kDa. e Protein levels of FHL2, normalized to calsequestrin levels and indexed to protein levels of healthy controls. Data are expressed as mean ± SEM. ***p < 0.001 vs. healthy controls, unpaired Student’s t test (in e). f–l Immunoelectron microscopy using an anti-FHL2 antibody in control and disease cardiac sections showing a specific labeling pattern in the I-band (zoom-in right upper corner of each image) of the sarcomeres; f human donor heart; g patient with HCM; h patient with aortic stenosis; i patient with obstructive HCM; j WT mouse; kMybpc3-targeted knock-in mouse. Scale bars 1 µm.  AoS aortic stenosis, Hom-KI homozygous Mybpc3-targeted knock-in mice, Het-KO heterozygous Mybpc3-targeted knock-out, Hom-KO homozygous Mybpc3-targeted knock-out mice, HOCM obstructive HCM
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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Fig1: FHL2 expression and sarcomeric localization in human and mouse hearts. FHL2 mRNA level in human (a) and mouse (b, c) HCM and normal hearts (n = 8 for human, n = 4–8 for mouse). Data are expressed as mean ± SEM. *p < 0.05 vs. healthy controls/WT, unpaired Student’s t test (in a and b) or one-way ANOVA with Dunnett’s multiple comparison vs WT (in c). d–e FHL2 protein level in human HCM and control hearts. d Representative Western blot stained with an antibody recognizing FHL2 and with an antibody directed against calsequestrin, used as an endogenous control. Molecular weight markers (MW) indicate 25 and 55 kDa. e Protein levels of FHL2, normalized to calsequestrin levels and indexed to protein levels of healthy controls. Data are expressed as mean ± SEM. ***p < 0.001 vs. healthy controls, unpaired Student’s t test (in e). f–l Immunoelectron microscopy using an anti-FHL2 antibody in control and disease cardiac sections showing a specific labeling pattern in the I-band (zoom-in right upper corner of each image) of the sarcomeres; f human donor heart; g patient with HCM; h patient with aortic stenosis; i patient with obstructive HCM; j WT mouse; kMybpc3-targeted knock-in mouse. Scale bars 1 µm.  AoS aortic stenosis, Hom-KI homozygous Mybpc3-targeted knock-in mice, Het-KO heterozygous Mybpc3-targeted knock-out, Hom-KO homozygous Mybpc3-targeted knock-out mice, HOCM obstructive HCM
Mentions: It has been shown that FHL2 protein level is markedly reduced in human failing hearts [4]. To examine whether FHL2 expression is altered in HCM we measured both mRNA and protein levels in ventricular tissue of HCM patients carrying MYBPC3 mutations [52]. FHL2 mRNA and FHL2 protein levels were >50 % lower in HCM patients than in controls (Fig. 1a, d, e). Similarly, FHL2 mRNA levels were markedly reduced in two HCM mouse models, the homozygous Mybpc3-targeted knock-in (Hom-KI) [59], and both heterozygous (Het-KO) and homozygous (Hom-KO) Mybpc3-targeted knock-out mice (Fig. 1b, c; [6]). Moreover, whereas no difference was observed in the right ventricle between WT, Het-KO and Hom-KO, FHL2 mRNA level was lower merely in the hypertrophied parts of the Het-KO (=septum) and Hom-KO (=septum + LV; Online Fig. 1).Fig. 1

Bottom Line: HCM is a myocardial disease characterized by left ventricular hypertrophy, diastolic dysfunction and increased interstitial fibrosis and is mainly caused by mutations in genes coding for sarcomeric proteins.We assessed the structural and functional consequences of the nonsynonymous substitutions after adeno-associated viral-mediated gene transfer in cardiac myocytes and in 3D-engineered heart tissue (EHT).Finally, chronic phenylephrine stimulation depressed EHT function in all groups, but to a lower extent in T171M-transduced EHTs.

View Article: PubMed Central - PubMed

Affiliation: Department of Experimental Pharmacology and Toxicology, Cardiovascular Research Center, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.

ABSTRACT
Based on evidence that FHL2 (four and a half LIM domains protein 2) negatively regulates cardiac hypertrophy we tested whether FHL2 altered expression or variants could be associated with hypertrophic cardiomyopathy (HCM). HCM is a myocardial disease characterized by left ventricular hypertrophy, diastolic dysfunction and increased interstitial fibrosis and is mainly caused by mutations in genes coding for sarcomeric proteins. FHL2 mRNA level, FHL2 protein level and I-band-binding density were lower in HCM patients than control individuals. Screening of 121 HCM patients without mutations in established disease genes identified 2 novel (T171M, V187L) and 4 known (R177Q, N226N, D268D, P273P) FHL2 variants in unrelated HCM families. We assessed the structural and functional consequences of the nonsynonymous substitutions after adeno-associated viral-mediated gene transfer in cardiac myocytes and in 3D-engineered heart tissue (EHT). Overexpression of FHL2 wild type or nonsynonymous substitutions in cardiac myocytes markedly down-regulated α-skeletal actin and partially blunted hypertrophy induced by phenylephrine or endothelin-1. After gene transfer in EHTs, force and velocity of both contraction and relaxation were higher with T171M and V187L FHL2 variants than wild type under basal conditions. Finally, chronic phenylephrine stimulation depressed EHT function in all groups, but to a lower extent in T171M-transduced EHTs. These data suggest that (1) FHL2 is down-regulated in HCM, (2) both FHL2 wild type and variants partially protected phenylephrine- or endothelin-1-induced hypertrophy in cardiac myocytes, and (3) FHL2 T171M and V187L nonsynonymous variants induced altered EHT contractility. These findings provide evidence that the 2 novel FHL2 variants could increase cardiac function in HCM.

Show MeSH
Related in: MedlinePlus